Method of stabilizing dye solutions and stabilized dye compositions

ABSTRACT

Stabilized metachromatic dyes, especially at application concentrations, comprising metachromatic dyes dissolved in one or more non-aqueous solvents, as well as metachromatic dyes dissolved in pH stabilized aqueous solutions. Also, various combinations of treatments are disclosed for stabilizing metachromatic dyes.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] The present invention relates to the stabilization ofmetachromatic dyes, and compositions comprising stabilized metachromaticdyes. The present invention is also directed to processes fordetermining polyionic materials utilizing stabilized metachromatic dyecompositions.

[0003] 2. Discussion of Background Information

[0004] Colorimetry is a well-known method of chemical analysis whichinvolves the comparison and matching of a standard color with that of anunknown color to approximate the concentration of a specific componentin a sample to be analyzed. When the amount of light absorbed by a givensubstance in solution is proportional to the concentration of theabsorbing species, colorimetry is a simple and accurate method fordetermining unknown concentrations. For example, if the concentration ofa polymer in an aqueous system is to be determined, a sample can betaken, the absorbance of the sample in the presence of a suitable dyecan then be measured and compared with a calibration curve to quicklyand accurately estimate the concentration of the polymer in the aqueoussystem. Colorimetry provides advantageous testing since it can easily beperformed at the application site.

[0005] Certain dyes undergo a unique color change upon interaction withpolyionic compounds in solution known as metachromasy. Thus,metachromatic dyes are those which undergo a color change uponinteraction with polyionic compounds. Any metachromatic dye can be usedin a colorimetric test to determine the concentration of a substance,including polycarboxylates, sulfonates, and the like in an aqueoussolution. More specifically, when anionic polymers contact ametachromatic dye, the dye molecules align with the anionic charges onthe polymers, resulting in a shift in the wavelength of maximumabsorbance of the dye molecule. This shift is observable as a colorchange in the solution containing the dye and the polymer. Thus, sincepolycarboxylates and sulfonates, which are anionic, induce ametachromatic change in certain dyes, their concentrations in aqueoussolutions can be determined colorimetrically by measuring theabsorbance, at a specified wavelength, of a solution containingpolycarboxylates and/or sulfonates and a metachromatic dye and comparingthis absorbance to absorbances of standards having known concentrationsof the species being measured.

[0006] However, when metachromatic dyes are dissolved in aqueoussolutions for use in analytical determinations, fresh samples must beprepared on a daily basis to insure accurate analysis. Aqueous solutionsof metachromatic dyes are extremely susceptible to degradation due to avariety of factors, such as light, temperature, dissolyed oxygen, pH,etc. For example, when pinacyanol chloride is dissolved in an aqueoussolution at a concentration of 9.0×10⁻⁵ molar, the pinacyanol chloridedegrades at a rate of approximately 10 to 20% per week. Because of thisdegradation problem, frequent reagent preparations must be made in thefield and this is not practical.

[0007] Still further, the instability of known dye solutions leads todisadvantageous results associated with the detection process. Thesedisadvantages result in a lack of reproducibility of results, i.e.,consistency of results is difficult to attain on separate days even withthe same water sample.

[0008] Accordingly, there is a need to provide stable metachromatic dyesthat enable simple tests for materials, particularly water treatmentpolymers, and especially enable simple tests that can be utilized overextended periods of time without the need for formulation in the field.

SUMMARY OF THE INVENTION

[0009] The present invention concerns methods for stabilizingmetachromatic dyes so that the dyes will not be susceptible todegradation and stabilized compositions comprising the metachromaticdyes. This will ensure that any colorimetry testing using the stabilizeddyes can be performed with the added assurance of reproducible results.

[0010] The present invention is directed to stabilized metachromatic dyecompositions, including aqueous solutions and non-aqueous solutions ofmetachromatic dye, having a percent change in absorbance of less thanabout 10% when stored for a period of about one week, more preferablyless than about 1% when stored for a period of about one week, even morepreferably less than about 3% when stored for a period of about onemonth, even more preferably less than about 5% when stored for a periodof about 3 months, even more preferably less than about 5% when storedfor a period of about 6 months, even more preferably less than about 10%when stored for a period of about one year, and even more preferablyless than about 5% when stored for a period of about one year.

[0011] Further, the present invention is directed to aqueous solutionsof metachromatic dye comprising metachromatic dye in an aqueous solvent,the aqueous solvent having a metachromatic dye stabilizing pH.

[0012] Still further, the present invention is directed to non-aqueoussolutions of metachromatic dye comprising metachromatic dye andnon-aqueous solvent, the non-aqueous solution being substantially freeof water.

[0013] The aqueous solution of metachromatic dye can comprise aqueoussolvents having a pH of a least about 8, more preferably a pH of a leastabout 10, and even more preferably a pH of a least about 11. PreferredpH ranges of the aqueous solvents include a pH range of about 8 to 14,even more preferably a pH range of about 11 to 12, and even morepreferably a pH range of about 11 to 11.5. The aqueous solution ofmetachromatic dye can include at least one basic material, such as abuffer, or a material such as at least one of sodium hydroxide,potassium hydroxide and lithium hydroxide, preferably sodium hydroxide.Moreover, the aqueous solution of metachromatic dye can include at leastone non-aqueous solvent.

[0014] The metachromatic dye can comprise at least one of pinacyanolchloride, crystal violet, methyl green, malachite green, acridin orange,paraosaniline, nile blue A, neutral red, safrin O, methylene blue,methyl red, brilliant green, toluidine blue, new methylene blue,quinalizarin, tetrachrome, brilliant blue G, and mordant black II, andis preferably pinacyanol chloride.

[0015] The non-aqueous solvent can comprise at least one of alcohols,methylcellosolve, hexane, pentane, heptane, toluene, xylene, benzene,dichlorobenzene, acetone, ethyl acetate, diethyl ether, acetonitrile,dimethylsulfoxide, preferably at least one of methanol, ethanol,butanol, isopropanol, propanol and ethylene glycol.

[0016] The non-aqueous solution of metachromatic dye can include one ormore non-aqueous solvents, preferably a mixture of methanol and ethyleneglycol having a preferred 25 vol % of methanol to 75 vol % of ethyleneglycol, with the non-aqueous solution preferably having a density at 25°C. of about 0.95 to 1.2 g/cm³, more preferably about 1 to 1.1 g/cm³, andeven more preferably about 1 to 1.05 g/cm³.

[0017] The non-aqueous solution of metachromatic dye is preferably freeof water.

[0018] The non-aqueous solvent preferably comprises high purity solvent.

[0019] The solutions of metachromatic dye preferably include at leastone oxygen scavenger.

[0020] The solutions of metachromatic dye preferably are in thesubstantial absence of oxygen.

[0021] The solutions of metachromatic dye preferably are purged with aninert gas.

[0022] The present invention is also directed to containers includingtherein the solutions of metachromatic dye according to the presentinvention. The container at least reduces the transmission of light, andpreferably prevents the transmission of light.

[0023] The present invention is also directed to methods of determiningmaterials, such as polyionic materials, preferably polyionic polymersutilizing the metachromatic dye solutions according to the presentinvention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The above and other features and advantages of the presentinvention will be made apparent from the following description of thepreferred embodiments, given as non-limiting examples, with reference tothe accompanying drawings, in which:

[0025]FIG. 1 depicts a calibration curve for HPS-I at 480 nm plottingabsorbance vs. HPS-I concentration; and

[0026]FIG. 2 depicts plots of absorbance vs. concentration for HPS-Iplotting absorbance vs.

[0027] HPS-I concentration.

DETAILED DESCRIPTION OF THE PRESENT INVENTION

[0028] The particulars shown herein are by way of example and forpurposes of illustrative discussion of embodiments of the presentinvention only and are presented in the cause of providing what isbelieved to be the most useful and readily understood description of theprinciples and conceptual aspects of the present invention. In thisregard, no attempt is made to show structural details of the presentinvention in more detail than is necessary for the fundamentalunderstanding of the present invention, the description making apparentto those skilled in the art how varying forms of the present inventionmay be embodied in practice.

[0029] Unless otherwise stated, all percentages, parts, ratios, etc.,are by weight.

[0030] Unless otherwise stated, a reference to a compound or componentincludes the compound or component by itself, as well as in combinationwith other compounds or components, such as mixtures of compounds.

[0031] Further, when an amount, concentration, or other value orparameter, is given as a list of upper preferable values and lowerpreferable values, this is to be understood as specifically disclosingall ranges formed from any pair of an upper preferred value and a lowerpreferred value, regardless whether ranges are separately disclosed.

[0032] The present invention is directed to stable compositions ofmetachromatic dyes. According to the present invention, themetachromatic dye composition is stable if the absorbance ofmetachromatic dye composition changes by less than about 10%, morepreferably less than about 5%, even more preferably less than about 3%,and most preferably less than about 1%, when stored for a period ofabout I week, more preferably about 1 month, even more preferably about3 months, even more preferably about 6 months, even more preferablyabout 7.5 months, even more preferably about 10 months, and even morepreferably about 1 year or more.

[0033] In order to determine the change in absorbance of themetachromatic dye composition, an initial absorbance of themetachromatic dye composition is determined 3 minutes after mixing ofthe metachromatic dye and the solvent at any wavelength in the visiblespectrum of 300 to 700 nm to obtain the absorbance data for theinitially prepared metachromatic dye composition. While one or morewavelengths can be utilized and/or measurements can be made over thewhole spectrum, preferably the wavelength of light is preferably at orabout the wavelength that provides maximum absorbance. The metachromaticdye composition, i.e., either the same dye composition that was testedto provide the initial absorbance, another portion of the samemetachromatic dye composition, or a metachromatic dye composition thathas been formulated to be identical to the metachromatic dye compositiontested for absorbance is then stored at ambient temperature, i.e., at25° C., for the period of time under dark conditions, such as by beingstored in an amber container, for which the stability test is to beperformed. Absorbance of the metachromatic dye composition is determinedat the same wavelength or wavelengths of light as for the initiallyprepared metachromatic composition. Calculations are then performed todetermine the percent change in absorbance.

[0034] Still further, the stabilized metachromatic dye compositionsaccording to the present invention are capable of use as metachromaticdyes in analytic determination of polyionic compounds, particularlypolyionic compounds including polycarboxylate and/or sulfonateconcentrations in aqueous systems.

[0035] The present invention is directed to any technique formaintaining the stability of the metachromatic dye composition, and avariety of techniques for providing stability to metachromatic dyecompositions are disclosed herein. Furthermore, it is noted thatstability techniques disclosed herewith can be utilized individually andin combination with each other.

[0036] The present invention is directed to any metachromatic dye thatcan be stabilized according to the present invention. In particular,metachromatic dyes that are most preferred for the present inventioninclude metachromatic dyes that are suitable for use in colorimetrictests for determining polyionic compounds in aqueous systems, preferablyfor determining polycarboxylate and/or sulfonate concentrations inaqueous systems. Examples of metachromatic dyes include, but are notlimited to, pinacyanol chloride, crystal violet, methyl green, malachitegreen, acridin orange, paraosaniline, nile blue A, neutral red, safrinO, methylene blue, methyl red, brilliant green, toluidine blue, newmethylene blue, quinalizarin, tetrachrome, brilliant blue G, and mordantblack II, preferably nile blue A and/or pinacyanol chloride.

[0037] The stability of the metachromatic dye composition can beenhanced by any technique that provides stability of the metachromaticdye composition. The following embodiments of the present invention aretherefore to be considered non-limiting embodiments setting forthpreferred manners of stabilizing metachromatic dye composition, andproviding guidelines in order that one having ordinary skill in the artcan provide, without undue experimentation, techniques for providingstabilized metachromatic dye compositions according to the presentinvention.

[0038] With the above in mind, it is noted that in one aspect thepresent invention achieves stabilized aqueous metachromatic dyecompositions by providing an aqueous composition of the dye having a pHof a least about 8, more preferably at least about 10, and even morepreferably at least about 11, with preferred pH ranges being from about8 to 14, more preferably from about 10 to 13, even more preferably fromabout 10.5 to 12.5, even more preferably about 11 to 12, and even morepreferably about 11 to 11.5, with one preferred pH value being about 11.For ease of reference the pH or pH range at which the metachromatic dyeis stabilized will be referred to herein as the metachromatic dyestabilizing pH. Thus, the terminology metachromatic dye stabilizing pHwill be utilized herein to denote a metachromatic dye composition havinga stabilizing pH in contrast to a non-stabilizing pH, such as an acidicpH.

[0039] The pH of the metachromatic dye composition can be adjustedand/or maintained in various manners to provide the metachromatic dyestabilizing pH without effecting or substantially effecting the coloringchanging or substantially changing the coloring changing ability of themetachromatic dye and the metachromatic dye composition, and preferablywithout changing or substantially changing the absorbance of themetachromatic dye and metachromatic dye composition. For example, basicmaterials including, but not limited to, at least one of sodiumhydroxide, lithium hydroxide, potassium hydroxide, rubidium hydroxide,calcium hydroxide, barium hydroxide, magnesium hydroxide and ammonia.

[0040] Preferably the basic material is at least one of sodiumhydroxide, such as a 1N solution of sodium hydroxide, lithium hydroxideand/or potassium hydroxide. Additionally, the pH of the metachromaticdye composition can be adjusted and/or maintained using buffer systemsthat provide the metachromatic dye stabilizing pH without affecting orsubstantially affecting the coloring changing or substantially changingthe coloring changing ability of the metachromatic dye and themetachromatic dye composition, and preferably without changing orsubstantially changing the absorbance of the metachromatic dye andmetachromatic dye composition. Preferably, the buffer system maintains apH of about 9 to 11 in the metachromatic dye composition. For example,buffer systems include, but are not limited to, potassiumcarbonate/potassium borate/potassium hydroxide (pH=10), boricacid/potassium chloride/sodium hydroxide (pH=9), sodiumhydroxide/glycine/sodium chloride (pH=11), sodium tetraborate (pH=9.18),and tris(hydroxymethyl)amino methane (pH=10.4).

[0041] The aqueous metachromatic dye compositions of the presentinvention can include other solvents therein in addition to water, suchas non-aqueous solvents. For examples, other solvents in addition towater can be included in the aqueous metachromatic dye compositions,such as, but not limited to, ethanol, methanol, butanol, propanol,ethylene glycol, methyl cellosolve, and glycol. For example, the volumeratio of water to other solvent can range from about 99 vol % to 1 vol%, more preferably from about 80 vol % to 20 vol %, even more preferablyabout 75 vol % to 25 vol %, even more preferably about 50 vol % to 50vol %, even more preferably from about 20 vol % to 80 vol %, and evenmore preferably from about 10 vol % to 90 vol %, and can even be as lowas 1 vol % to 99 vol %.

[0042] When the aqueous metachromatic dye composition is utilized in atest the pH of aqueous metachromatic dye composition is preferablyadjusted to be at a neutral pH of about 6.5 to 7. Adjustment of the pHcan be affected in any manner that lowers the pH while not interferingwith the metachromatic dye, such as by adding an acid, such as, but notlimited to, sulfuric acid, nitric acid, or a buffer, such as, but notlimited to, meta buffer, i.e., 10.1 wt % EDTA (ethylendiaminetetraaceticacid) tetrasodium salt, 11 wt % potassium phosphate monobasic, and 78.9wt % distilled water.

[0043] In another aspect, the present invention achieves stabilizedmetachromatic dye compositions by providing non-aqueous compositions ofthe dye. In this regard, it has been discovered that the stability ofmetachromatic dyes is deteriorated by its inclusion in an aqueousenvironment. In particular, it is noted that when metachromatic dyes arestored in non-aqueous compositions stable metachromatic dye compositionsare obtained.

[0044] The non-aqueous metachromatic dye compositions according to thepresent invention can include water therein, such as trace amounts ofwater that do not affect the stability of the metachromatic dyecompositions. For ease of reference, these compositions are referred toherein as non-aqueous metachromatic dye compositions or solutions. Inother words, the non-aqueous metachromatic dye compositions of thepresent invention can include amounts of water therein whereby thecompositions are in conformance with the stability requirements of thepresent invention. Thus, when referring to a non-aqueous compositionthat is substantially free of water, such composition is intended toinclude up to an amount of water whereby the composition conforms withthe stability requirements of the present invention. Preferably, thenon-aqueous metachromatic dye compositions of the present inventioncontain, at most, trace amounts of water, and preferably do not includewater therein.

[0045] The non-aqueous metachromatic dye composition can be formulatedusing a variety of non-aqueous solvents in which the one or moremetachromatic dyes intended to be included in the metachromatic dyecomposition are soluble. Therefore, in order to determine whether anon-aqueous solvent is a non-aqueous solvent or mixture of non-aqueoussolvents that is utilizable in the non-aqueous metachromatic dyecomposition according to the present invention, a non-aqueous solvent ormixture of the non-aqueous solvents should be mixed with the one or moremetachromatic dyes that are intended to be utilized in the non-aqueousmetachromatic dye composition to determine if the one or moremetachromatic dyes are soluble in the non-aqueous solvent or mixture ofnon-aqueous solvents. If the one or more metachromatic dyes are solublein the non-aqueous solvent or mixture of non-aqueous solvents, thenon-aqueous metachromatic dye composition can be formulated with thenon-aqueous solvent or mixture of non-aqueous solvents and subjected tothe herein discussed stabilization test to determine whetherstabilization is achieved for the non-aqueous solvent or mixture ofnon-aqueous solvents.

[0046] The non-aqueous solvents can include, but are not limited to,alcohols such as methanol, ethanol, butanol, propanol, isopropanol,ethylene glycol, propylene glycol, glycerin (glycerol); and organicsolvents such as methylcellosolve, hexane, pentane, heptane, toluene,xylene, benzene, -dichlorobenzene, acetone, ethyl acetate, diethylether, acetonitrile, dimethylsulfoxide. Preferred solvents includemethanol, ethanol, isopropanol, butanol, propanol and ethylene glycol.

[0047] Preferably, the non-aqueous solvent has a density at 25° C. ofabout 0.95 to 1.2 g/cm³, more preferably 1 to 1.1 g/cm³, and even morepreferably 1 to 1 .05 g/cm³, with a preferred value of density beingabout 1 g/cm³. To achieve this preferred density, the non-aqueoussolvent can be composed of one non-aqueous solvent that has a densitywithin the preferred range, or a mixture of non-aqueous solvents, withone, some or all of the non-aqueous solvents within the mixturecomprising a density that is not within the preferred density range;however, the density of the mixture of non-aqueous solvents willpreferably be within the preferred range.

[0048] Expanding upon the above, it is noted that single non-aqueoussolvent or mixtures of non-aqueous solvents can be utilized in thenon-aqueous metachromatic dye compositions of the present invention. Inthis regard, it is noted that the viscosities of non-aqueous solventscan be lower than desired, whereby non-aqueous metachromatic dyecompositions incorporating these non-aqueous solvents can be toovolatile. Still further, it is noted that the viscosities of othernon-aqueous solvents can be higher than desired, whereby non-aqueousmetachromatic dye compositions incorporating these non-aqueous solventscan be too viscous. Therefore, it is preferable that the non-aqueoussolvent have a density at 25° C. of about 0.95 to 1.2 g/cm³, morepreferably 1 to 1.1 g/cm³, and even more preferably 1 to 1.05 g/cm³,with a preferred value being about 1 g/cm³, or comprise mixtures ofnon-aqueous solvents, which mixture has a density at 25° C. of about0.95 to 1.2 g/cm³, more preferably 1 to 1.1 g/cm³, and even morepreferably 1 to 1.05 g/cm³, with a preferred value being about 1 g/cm³.

[0049] The mixture of non-aqueous solvents of differing densities cancomprise various mixtures of non-aqueous solvents, with one preferredmixture being composed of methanol (density of 0.8 g/cm³ at 25° C.) andethylene glycol (density of 1.1 g/cm³ at 25° C.). Preferably the higherdensity non-aqueous solvent is present in the mixture in a range ofabout 50 vol % to 95 vol %, with the lower density non-aqueous solventbeing present in the mixture in a range of about 5 vol % to 50 vol %.Particularly preferred values include wherein the higher densitynon-aqueous solvent is present in the mixture at about 60 vol %, withthe lower density non-aqueous solvent being present in the mixture atabout 40 vol %; wherein the higher density non-aqueous solvent ispresent in the mixture at about 70 vol %, with the lower densitynon-aqueous solvent being present in the mixture at about 30 vol %; andwherein the higher density non-aqueous solvent is present in the mixtureat about 75 vol %, with the lower density non-aqueous solvent beingpresent in the mixture at about 25 vol %. In other words, it ispreferred that the volume ratio of higher density non-aqueous solvent tolower density non-aqueous solvent is about 19:1 to 1:1, more preferablyabout 3:1 to 1.5:1, and even more preferably about 3:1 to 2.3:1. Aparticularly preferred non-aqueous solvent mixture comprises methanolpresent in an amount of about 25 volume percent and ethylene glycolpresent in an amount of about 75 volume percent.

[0050] Further beneficial improvements to the metachromatic dyecompositions according to the present invention can be achieved byutilizing high purity materials, such as distilled water in the case ofaqueous metachromatic dye compositions, such as high purity non-aqueoussolvents and high purity viscosity increasing agent or diluents in thecase of a non-aqueous metachromatic dye composition.

[0051] In the case of non-aqueous solvents, high purity is referred toherein as a purity of at least 99 wt %. It is noted that high purity isusually associated with technical grade materials, as compared toreagent grade materials.

[0052] The higher purity materials provide metachromatic dyecompositions according to the present invention with at least lowerconcentrations of interfering agents. For example, low purity materialscan add interfering agents, such as iron, calcium and/or magnesium tothe metachromatic dye composition, which interfering agents caninterference with both the metachromatic dye and the analytical test,such as an analytical test for polyionic polymers.

[0053] Further beneficial improvements to the metachromatic dyecompositions according to the present invention can be achieved bystoring the metachromatic dye composition in an oxygen free orsubstantially oxygen free environment. The oxygen free or substantiallyoxygen free environment can be obtained in any manner by which oxygen,such as molecular oxygen, or oxidative components in the composition areavoided and/or removed from the metachromatic dye composition. Forexample, and without limiting the present invention, an oxygen free orsubstantially oxygen free environment can be achieved by utilizing oneor more of a variety of techniques including storing the metachromaticdye composition in a sealed container, purging the metachromatic dyecomposition with an inert gas, such as argon or nitrogen, utilizing anoxygen scavenger, such as, but not limited to, sodium sulfite, sodiumbisulfite, ascorbate, hydrazine, hydroquinone, benzohydroquinone, orsealing the metachromatic dye composition under vacuum or partialvacuum.

[0054] With regard to sealing the metachromatic dye composition in acontainer, it is noted that the metachromatic dye composition can besealed in any container that limits diffusion of oxygen into themetachromatic dye composition. Accordingly, the metachromatic dyecomposition can be contained in a container that is sealed, such as byutilizing a closure element that can seal an opening in the container.For example, and without limiting the invention, the closure element caninclude a cap, such as a cap that can be screwed over the opening of thecontainer to seal the opening, a cap that can be sealingly pressedagainst the opening to obtain a seal between the opening and the cap; ora cork type element, such as a rubber cork that can be inserted into theopening. A particularly preferred container is an 8 oz. Amber BostonRound, 24/400 finish (referring to the threads on neck of bottles)including a 24/400 black phenolic polyseal cone liner as a cap, asdistributed by PENN Bottle and Supply Co., Philadelphia, Pa., with theamber container being manufactured by Lawson Mardon Wheaton, Millbille,N.J., and the cap being manufactured by Poly/Seal Corp., Baltimore, Md.

[0055] Still further, the containers can comprise completely sealedcontainers, such as ampoules. In such types of containers, it is oftennecessary that the container be broken to enable release of thecomposition contained therein.

[0056] With respect to the use of oxygen scavengers, the oxygenscavenger can comprise inorganic and/or organic materials, such as, butnot limited to, sodium sulfite, sodium bisulfite, ascorbate, hydrazine,hydroquinone, benzohydroquinone. The oxygen scavenger is preferablyadded to the metachromatic dye composition during its preparation, or assoon as possible after the metachromatic dye composition is prepared. Inthe instance where the oxygen scavenger is added after preparation ofthe metachromatic dye composition, it is preferred that the oxygenscavenger be added to the metachromatic dye composition immediatelyafter its production. Most preferably, the oxygen scavenger is added tothe solvent prior to the addition of the metachromatic dye.

[0057] With respect to purging with an inert gas, any purging techniquecan be utilized to remove oxygen from the metachromatic dye composition.In this regard, without limiting the invention, the inert gas, such asnitrogen and/or argon, can be caused to bubble through or pass over themetachromatic dye composition, preferably passed over the metachromaticdye composition. Of importance is that the purging technique issufficient to remove an effective amount of oxygen to enhance stabilityof the metachromatic dye composition. Moreover, the purging technique ispreferably adapted to remove as little of the solvent as possible,particularly in the case where the solvents are highly volatile. In oneexemplary purging technique, a piece of flexible tubing, such as Tygontubing, is connected to an inert gas tank, such as an argon tank, on oneend, and a glass or plastic pipette on the other end.

[0058] The inert gas tank is turned on, and the gas is gently bubbledthrough the solution in a container, such as a beaker, an Erlenmeyerflask, etc.

[0059] Similarly, any technique for sealing a metachromatic dyecomposition under vacuum or partial vacuum can be utilized. Thus, forexample, the metachromatic dye composition can be placed in a container,and a vacuum pulled on the container and its content to achieve a vacuumof less than about 100 mm of mercury (1 ppm O₂), more preferably lessthan least about 50 mm of mercury (0.5 ppm O₂), and even more preferablyless than about 20 mm of mercury (0.2 ppm O₂).

[0060] Moreover, further beneficial improvements to the metachromaticdye compositions according to the present invention can be achieved bystoring the metachromatic dye composition in a container that protectsthe metachromatic dye composition from light, especially ultravioletradiation. Thus, any container that at least partially limits, andpreferably completely blocks, the entrance of light into themetachromatic dye composition, can be preferably utilized to store themetachromatic dye composition. For example, the walls of the containercan be constructed from an opaque material that will completely blockthe entrance of light in the container and/or the container can beplaced in a dark environment, such as a cardboard carton or a styrenepackage. Still further, the walls of the container can be constructedfrom a translucent material that permits light to partially enter themetachromatic dye composition.

[0061] As examples of materials of construction for the container,without limiting the invention, the container can be constructed ofglass that is treated and/or colored to prevent or limit penetration oflight into the container, such as amber colored glass, plasticcontaining materials to block or limit the entrance of light, such asmaterials that will at least limit or block the passage of ultravioletradiation, or a metal. A preferred amber bottle is obtainable fromNalgene International, and is constructed from HDPE. An opaque containerthat prevents light transmission can be constructed of Teflonfluorinated ethylene propylene (FEP), which is used for extraordinarychemical resistance. Also a black Teflon FEP container containing acarbon black pigment for zero transmission of both visible andultraviolet light is also available from Nalgene International. If thecontainer is constructed from materials that may be an interferant tothe metachromatic dye or materials to be analyzed, it is preferred toinclude a liner and/or a coating, such as a liner on a cap, or a coatingon internal and/or external walls of the container. In particular,liners typically refer to inserts for the caps or closures on thebottles. For example, caps with flat, disc-type polyvinyl liners, capswith flat, disc-type Teflon TPE fluorocarbon resin liners, polypropylenefilm foam liners, black phenolic rubber-lined closures, and a preferredliner comprising black phenolic caps with conical low-densitypolyethylene Poly-Seal liners that form especially good seals. The capsor closures,can be constructed of the same materials as the containers,for example, amber propylene or polypropylene screw closures,polyethylene caps and/or Nalgene International TEFZEL®(ethylene-tetrafluoroethylene) closures. Coatings are typically used inreference to the outside of the container, and can include, for example,polyvinyl chloride, which is commonly used in coating acid bottles tohelp prevent breakage and spills. Moreover, a container, such as a clearcontainer, can be coated with aluminum foil, Styrofoam, etc.

[0062] Moreover, the container can be constructed from a material thatis transparent to light if the container is stored in another containerthat blocks or limits the passage of light. For example, the material ofconstruction of the container can be clear glass or plastic, and, asdiscussed above, the container can be placed in a dark environment, suchas a cardboard carton or a styrene package, or have a liner positionedin the container and/or a coating placed exteriorly of the container toblock the transmission of light.

[0063] Materials of construction for a clear container can be glassand/or a resin, such as high density polyethylene (HDPE), low densitypolyethylene (LDPE), polycarbonate (PC), polyvinyl chloride (PVC),polypropylene (PP) and/or polymethylpentene (PMP). If the container isconstructed from these clear materials, preferably direct UV exposureshould be avoided, with the clear container being modified and/or placedin a dark environment.

[0064] However, preferably, the container is constructed from a materialthat limits or blocks the passage of light into the container.

[0065] Still further, beneficial improvements to the metachromatic dyecompositions according to the present invention can be achieved bystoring the metachromatic dye composition under low temperatureconditions. In particular, the metachromatic dye composition can bestored at temperature lower than about 15° C., more preferably lowerthan about 10° C., even more preferably lower than about 8° C. Themetachromatic dye composition is preferably stored at temperatures highenough so as not to freeze the metachromatic dye composition. Preferredtemperature ranges are about 15° C. to 3° C., more preferably about 10°C. to 4° C., more preferably about 8° C. to 4° C., and most preferablyabout 6° C. to 4° C.

[0066] It is noted that the herein described techniques forstabilization of the metachromatic dye compositions can be utilized incombination with each other, and such combinations of techniques canprovide enhancement of the stabilization. Thus, for example, anon-aqueous metachromatic dye composition can be stored in a containerthat limits or blocks light penetration into the bottle, with or withoutany combination of techniques for removal of oxygen from the non-aqueousmetachromatic dye composition, such as any combination of purging and/oruse of oxygen scavengers, with or without lower storage temperatures.Moreover, the aqueous metachromatic dye composition can be adjusted toan alkaline pH with or without storage under dark conditions, and withor without storage at lower temperatures.

[0067] The concentration of the metachromatic dye in the solvent ispreferably a concentration of the metachromatic dye that is in excess ofthat which is expected to react with the analyte to be determined, suchas a polyionic polymer. For example, for the polyionic polymer, HPS-I,obtained from BetzDearborn, Inc., Trevose, Pa., at a concentration ofabout 0.5 ppm of polymer in the sample, the metachromatic dyecomposition preferably has a metachromatic dye molar concentration of atleast about 9×10⁻⁵M.

[0068] It is noted that one mole of metachromatic dye reacts with 0.05moles SO₄ ⁻ (mole ratio of 1:0.05), or 0.15 moles of COO⁻ (mole ratio of1:0.15), or one mole of metachromatic dye to 10⁻³ moles HPS-I (moleratio of 1:0.001). Thus, the mole ratio of metachromatic dye moleculesto SO₄ ⁻ ranges from about 0.05 to 100, and the mole ratio ofmetachromatic dye molecules to COO— ranges from about 0.15 to 100. It istherefore preferred that the mole ratio of metachromatic dye topolycarboxylate and/or sulfonate groups is about 0.1 to 100, morepreferably about 1 to 50, and even more preferably about 1 to 30.

[0069] The concentration of the metachromatic dye in the metachromaticdye composition is optimized so the maximum metachromatic absorbance isobtained for a polyionic material to be assayed by the followingtechnique. Different concentrations of metachromatic dye composition arereacted with known concentrations of the polyionic material to beassayed, such as HPS-I, and a plot of absorbance vs. concentration aspolyionic material is plotted.

[0070] The slope of the curve is determined, and the optimummetachromatic dye composition includes a concentration of metachromaticdye, or a concentration range of metachromatic dye that provides thelargest slope with a small or smallest intercept. In this manner, themetachromatic dye composition is optimized so that a small change in theconcentration of the polyionic material to be assayed corresponds to alarge change in absorbance over the operable absorbance range of themetachromatic dye.

[0071] A particularly preferred metachromatic dye composition isformulated by mixing 0.0844 g of metachromatic dye, e.g., pinacyanolchloride, with 250 mL of non-aqueous solvent, e.g., methanol, and addingthe resulting mixture to 750 mL of viscosity increasing agent, e.g.,ethylene glycol, and mixing for a sufficient amount of time tohomogenize the solution, such as 30 minutes. This preferably provides apreferred composition of 25 vol % methanol and 75 vol % ethylene glycolwith 0.0844 grams of pinacyanol chloride

[0072] With respect to the formulation of the metachromatic dyecompositions, in the case of a non-aqueous solvent system, the dye canbe mixed with one or more solvents and/or one or more viscosityincreasing agents. When mixed with plural solvent/viscosity materials,the metachromatic dye can be individually mixed with one or more of thematerials, and subsequently mixed with any other materials in any order,or can be mixed with the total combination of materials. However, it ispreferred to mix the metachromatic dye with one of the more solublematerials, most preferably the most soluble material, and then mix theresulting composition with the other solvent materials.

[0073] When mixed with solvents that do not as easily solubilize themetachromatic dye, it is preferred that the resulting composition bemixed for a sufficient period of time so that the solution is clear inthat floating matter is not visible. For example, the mixing could beperformed for, but not limited to, about 30 minutes, to homogenize themetachromatic dye in the solvent system.

[0074] Preferably, a dye such as pinacyanol chloride is dissolved in anoxygen-free solvent and is packaged in glass (amber) ampoules. Thepackaging may be sealed under vacuum for maximum stability. Storageunder these conditions provides a dye with suitable stability bypreventing oxygen and/or light from contacting the dye solution.

[0075] The metachromatic dye compositions of the present invention canbe utilized in the determination of polyionic substances, particularly,polyionic polymers, in various environments, including, but not limitedto, in aqueous environments, such as potable water, industrial systems,cooling waters, boiler systems, industrial processes, and water andwaste water applications. For example, the metachromatic dyecompositions can be utilized to determine polycarbonates and sulfonatesdisclosed in U.S. Pat. No. 4,894,346 to Myers, which is incorporated byreference hereto in its entirety. Moreover, the metachromatic dyecompositions of the present invention can be utilized to monitor polymerconcentrations in cooling water field samples, such as to monitor HPS-I(acrylic acid/1-allyloxy, 2-hydroxypropylsulfonate), such as disclosedin U.S. Pat. No. 4,659,481 to Chen, which is incorporated by referencehereto in its entirety, as well as PESA (polyepoxysuccinic acid disodiumsalt), such as disclosed in U.S. Pat. No. 5,062,962 to Brown et al.,which is incorporated by reference hereto in its entirety.

[0076] To be utilizable as a metachromatic dye for analytical purposes,the metachromatic dye should be able to pass a two part test. In thefirst test, which is a quality of the metachromatic dye test, themetachromatic dye is mixed one part by volume with 50 parts of distilledwater. The resulting dye solution should provide a visible absorbancewithin 1.000±0.100 AU (Absorbance Units) utilizing a 1 inch (2.54 cm)path length when measured in a spectrophotometer at 600 nm, threeminutes after production of the metachromatic dye solution.

[0077] If a metachromatic dye passes the first test, it is subjected tothe second test. In the second test, a 1 ppm solution of polymer indistilled water is prepared by mixing 100 ml of 1 ppm standard solutionof polymer in distilled water 2 ml of buffer, preferably meta buffer(10.1 wt % EDTA (ethylendiaminetetraacetic acid) tetrasodium salt, 11 wt% potassium phosphate monobasic, and 78.9 wt % distilled water) and 5 mlof metachromatic dye. The absorbance of this solution is measured andcompared to a standard calibration curve utilizing a standard I ppmsolution of polymer in distilled water, such as illustrated in FIG. 1and tabulated in Table 3. The measured absorbance is read against thecalibration curve to determine the concentration of polymer. Theconcentration of polymer should be between 90% and 110% of the 1 ppmconcentration. For example, in the illustrated embodiment of FIG. 1 andTable 3, the concentration should be between 0.9 and 1.1 for themeasured absorbance for the metachromatic dye to pass the second test.

[0078] The determination of polyionic substances can be performedutilizing various techniques, and the following non-limiting techniquesare described to provide examples of both off-line and on-line methodsof performing the determination. For example, the off-line determinationof HPS-I can be accomplished by diluting the sample to obtain a 0.1 to1.5 ppm concentration of HPS-I, of which 100 ml is mixed with 2 ml of abuffer, such as meta buffer, and 5 ml of metachromatic dye composition.The mixture is preferably swirled for about 10 seconds, and at 45 to 50seconds, 25 ml is preferably transferred to the sample cell. At as closeto possible to the desired test time, such as 60 seconds, the absorbancemeasurement is performed, such as preferably at 480 nm in the case ofHPS-I.

[0079] It is noted that glassware and sample cell are preferably rinsedwith methanol after each use to avoid dye staining, then thoroughlyrinsed with distilled water. Moreover, the use of disposable pipettes ispreferred, as it is hard to clean and reuse pipettes.

[0080] The on-line determination of polyionic substances, such as HPS-I,can be achieved utilizing a ChemScan Analyzer, Model UV-6101,manufactured by Applied Spectrometry Associates, Inc. (ASA), Waukesha,Wis. 53186. This model allows the user to program a “read” sequence thatcan include up to twenty different mechanical, optical and mathematicalfunctions. The sample volume is preferably about 10 ml, and thepathlength is preferably 0.5 inch (1.27 cm). In the test, the flow cellis flushed and filled with a sample and the cell is air purged for 5seconds to create a small headroom for reagent additions. A buffer suchas 0.5 ml of meta buffer is injected and mixed for 5 seconds beforeinjecting 1 ml of the dye. The dye is mixed for 5 seconds and a visibleabsorbance scan between 400 and 650 nm is made after 20 seconds ofreaction time. A multi-wavelength chemometric calibration is applied tothe spectrum to determine the polymer concentration in the sample.

[0081] The invention will now be described with respect to certainexamples which are merely representative of the invention and should notbe construed as limiting thereof.

EXAMPLES

[0082] The invention is illustrated in the following non-limitingexamples, which are provided for the purpose of representation, and arenot to be construed as limiting the scope of the invention. All partsand percentages in the examples are by weight unless indicatedotherwise.

Examples 1-8

[0083] The stability of pinacyanol chloride, obtained from AldrichChemical Co., Milwaukee, Wis., in different solvent systems is tested,with all conditions being at room temperature, using a 1 inch (2.54 cm)pathlength cuvette, by dissolving the pinacyanol chloride in eachsolvent system, and recording the absorbance at 600 nm in a DR2000/2010Spectrophotometer supplied by HACH Company, Loveland, Colo., after threeminutes mixing time. It is noted that where less than 100 vol %indicated for a solvent system, the balance was distilled water.Moreover, solubility issues led to the use of different concentrationsof the dye in the different solutions. The concentrations and resultsare illustrated in Table 1, with storage of the dye solutions in amberbottles being in air for the time indicated in Table 1. Each absorbancewas measured for a minimum of 6 samples, and the average value isillustrated in Table 1. The % change from day 1 shown in the Table 1,and the other tables which follow is the change of the final measuredvalue as compared to the initial measurement.

[0084] The results illustrated in Table 1 demonstrate that Examples 2(100 vol % ethanol), 3 (50 vol % ethylene glycol in distilled water), 5(100 vol % methanol) and 8 (100 vol % Methyl Cellosolve) produced themost stable dye solution. TABLE 1 Stability of Pinacyanol Chloride inSolvent Systems as a function of time in days Ex. No. 1 2 3 4 5 6 7 8Solvent Ethylene Methyl Methyl Methyl Distilled Ethanol Glycol MethanolMethanol Cellosolve Cellosolve Cellosolve Water (100%) (50%) (5%) (100%)(2%) (5%) (100%) Dye Conc. 8.84 × 1.1 × 9.2 × 8.93 × 1.79 × 9.0 × 9.0 ×1.8 × 10⁻⁵M 10⁻⁵M 10⁻⁵M 10⁻⁵M 10⁻⁵M 10⁻⁵M 10⁻⁵M 10⁻⁵M Time (days)Absorbance at 600 nm 1 1.370 1.058 1.437 1.446 1.446 1.445 1.445 1.445 21.318 3 1.289 4 1.250 5 1.242 6 1.229 1.418 1.364 1.365 1.419 20 1.42135 1.428 58 1.464 82 1.057 % −9.3 −0.1 −0.6 −15.0 1.2 −5.6 −5.5 −1.7Change from day 1

Examples 9-11

[0085] 0.0844 g pinacyanol chloride is dissolved in a solvent system of250 milliliters (ml) of technical grade methanol obtained from CoyneChemical, Croydon, Pa., and 750 ml of ethylene glycol, obtained fromFischer Scientific, Pittsburgh, Pa. (Laboratory Grade indicated to betypically >99% pure), for a total volume of 1 liter. The solutions werestored at three temperatures of 4° C., 20° C. and 40° C. for the timeperiods indicated in Table 2. Storage of the samples and the absorbancemeasurements were made in a similar manner as set forth in Example 1except all experiments were run a minimum of three times with theaverage shown in Table 2. These examples show that metachromatic dyecompositions according to the present re stable for lengthy periods,including about 7.5 months. TABLE 2 Stability of Pinacyanol Chloride in25 vol %/75 vol % Methanol/Ethylene Glycol (1:3) Stored at DifferentTemperatures Ex. No. 9 10 11 Temperature 40° C. 20° C. 4° C. TimeAbsorbance at 600 nm (days) 1 1.011 1.011 1.011 48 1.025 1.040 1.014 551.021 1.028 1.051 83 1.079 1.090 1.087 123 0.948 0.889 0.973 166 1.0270.995 1.083 194 0.989 0.989 1.094 230 0.977 0.942 1.077 % Change −3.4−6.8 6.5 from day 1

Example 12

[0086] A calibration curve for HPS-I, obtained from BetzDearbornDivision of Hercules incorporated Trevose PA, was prepared by reactingdifferent concentrations of the HPS-I dissolve in distilled water mixedwith 2 ml of META buffer and 5 ml of the pinacyanol chloride solution inmethanol/ethylene glycol of Examples 9-11 except that the methanol isCertified A.C.S Grade Methanol with a purity of 99.8% obtained fromFischer Scientific, Pittsburgh Pa. The visible absorbance absorbance at480 nm was measured after reacting for 1 minute using a DR2000/2010Spectrophotometer available from HACH Company, Loveland, Colo. A 1-inch(2.54 pathlength cuvette holding a total volume of 25 ml was utilized,and the dye solutions were stored in amber bottles in air for theduration of all experiments. Each absorbance was measured for a minimumof 3 samples, and the average value is illustrated in Table 3, and theresulting calibration curve is shown in FIG. 1. TABLE 3 Concentration(ppm) of HPS-I Absorbance @ 480 nm 0 0.157 0.1 0.213 0.2 0.267 0.5 0.4031.0 0.582 1.5 0.696

Examples 13-27

[0087] The pinacyanol chloride solution in a 25:75 vol % of methanol toethylene glycol of Examples 9-11 was stored for varying period of times.The stored pinacyanol chloride solutions were prepared as in Example 12,and then reacted with the same known concentration of HPS-I, and thevisible absorbance was measured in the same manner as in Example 12.

[0088] The solution having a known concentration of HPS-I was preparedby mixing HPS-I in distilled water to give a concentration of 1 ppmHPS-I to achieve a test solution that provides an absorbance readingwithin the calibration range of 0-1.5 ppm, as illustrated in FIG. 1. Theresults are shown in Table 4. TABLE 4 Concentration of HPS-I Recoveredas a Function of Time (in days) Using Pinacyanol Chloride in 25 Vol %MeOH:75 Vol % Ethylene Glycol. Example Time Temperature AbsorbanceConcentration No. (Days) (° C.) (@ 480 nm) Recovered, ppm 13  83 400.605 1.07 14  83 20 .0594 1.03 15  83  4 0.607 1.07 16 123 40 0.5911.02 17 123 20 0.584 1.00 18 123  4 0.588 1.01 19 166 40 0.593 1.03 20166 20 0.579 0.99 21 166  4 0.603 1.07 22 194 40 0.594 1.03 23 194 200.575 0.97 24 194  4 0.604 1.07 25 230 40 0.590 1.01 26 230 20 0.5720.96 27 230  4 0.595 1.03

Examples 28-56

[0089] The stability of 9×10⁻⁵ pinacyanol chloride, obtained fromAldrich Chemical Co., Milwaukee, Wis., is tested in aqueous media asshown in Tables 5-14, with all conditions being at the room temperature,using a 1 inch (2.54 cm) pathlength cuvette, by dissolving thepinacyanol chloride in an aqueous system, and recording the absorbanceat 600 nm in the above-noted DR2000/2010 Spectrophotometer after a 3minute mixing time. Storage of the dye solutions is in amber bottles inair for the time indicated in the tables. Each absorbance was measuredin at least duplicate, and the average value is illustrated in thetables.

[0090] Table 5 shows the stability of pinacyanol chloride in thepresence of strong base.

[0091] Table 6 shows the stability of pinacyanol chloride in thepresence of oxygen scavenger.

[0092] Table 7 shows the stability of pinacyanol chloride in thepresence of oxygen scavenger.

[0093] Table 8 shows the stability of pinacyanol chloride in thepresence of strong based and oxygen scavenger.

[0094] Table 9 shows the stability of pinacyanol chloride in thepresence of oxygen scavenger.

[0095] Table 10 shows the stability of pinacyanol chloride in thepresence of oxygen scavenger.

[0096] Table 11 shows the stability of pinacyanol chloride in thepresence of strong base and oxygen scavenger.

[0097] Table 12 shows the stability of pinacyanol chloride in thepresence of buffer.

[0098] Table 13 shows the stability of pinacyanol chloride in thepresence of strong acid.

[0099] Table 14 shows the stability of pinacyanol chloride in thepresence of oxygen scavenger.

[0100] As can be seen from the results depicted in these Tables 5-14,the presence of a strong acid or oxygen scavenger per se, the dyediscolors quickly. In the presence of 0.15 wt/wt of NaOH, the dye fadingis minimal (˜3% in 6 days). TABLE 5 Stability of Pinacyanol Chloride inthe Presence of Strong Base % Change Ex. Time Absorbance from No. (days)System @ 600 nm Day 1 Observations 28 1 Distilled Water 1.196 DarkPurple, (DI) + No Change 1 ml NaOH (1N) 29 2 DI + 1.213 1.4 Dark Purple,No Change 1 ml NaOH (1N) 30 6 DI + 1.164 −2.7 Dark Purple, No Change 1ml NaOH (1N)

[0101] TABLE 6 Stability of Pinacyanol Chloride in the Presence ofOxygen Scavenger % Change Ex. Time Absorbance from No. (days) System @600 nm Day 1 Observations 31 1 DI + 0.1 gm 1.089 Sky Blue, Na₂SO₃Significant Color Change 32 2 DI + 0.1 gm 1.14 4.7 Sky Blue, Na₂SO₃Significant Color Change 33 6 DI + 0.1 gm 0.741 −32 Sky Blue, Na₂SO₃Significant Color Change

[0102] TABLE 7 Stability of Pinacyanol Chloride in the Presence ofOxygen Scavenger % Change Ex. Time Absorbance from No. (days) System @600 nm Day 1 Observations 34 1 DI + 0.2 gm 1.03 Sky Blue, Na₂SO₃Significant Color Change 35 2 DI + 0.2 gm 1.12 −88.3 Sky Blue, Na₂SO₃Significant Color Change 36 6 DI + 0.2 gm 0.557 −44 Pre- Na₂SO₃cipitation

[0103] TABLE 8 Stability of Pinacyanol Chloride in the Presence ofStrong Base and Oxygen Scavenger % Change Ex. Time Absorbance from No.(days) System @ 600 nm Day 1 Observations 37 1 DI + 0.1 gm 1.089 SkyBlue, Na₂SO₃ +1 mL Significant NaOH (1N) Color Change 38 2 DI + 0.1 gm1.14 4.7 Sky Blue, Na₂SO₃ +1 mL Significant NaOH (1N) Color Change 39 6DI + 0.1 gm 0.741 −32 Sky Blue, Na₂SO₃ +1 mL Significant NaOH (1N) ColorChange

[0104] TABLE 9 Stability of Pinacyanol Chloride in the Presence ofOxygen Scavenger % Change Ex. Time Absorbance from No. (days) System @600 nm Day 1 Observations 40 1 DI + 0.1 gm 0.884 Sky Blue, Na₂SO₃Significant Color Change 41 2 DI + 0.1 gm 0.719 −18.7 Sky Blue, Na₂SO₃Significant Color Change 42 6 DI + 0.1 gm 0.713 −19.3 Pre- Na₂SO₃cipitation

[0105] TABLE 10 Stability of Pinacyanol Chloride in the Presence ofOxygen Scavenger % Change Ex. Time Absorbance from No. (days) System @600 nm Day 1 Observations 43 1 DI + 0.2 gm 1.16 Sky Blue, Na₂SO₃Significant Color Change 44 2 DI + 0.2 gm 1.117 −3.7 Sky Blue, Na₂SO₃Significant Color Change 45 6 DI + 0.2 gm 0.871 −25 Pre- Na₂SO₃cipitation

[0106] TABLE 11 Stability of Pinacyanol Chloride in the Presence ofStrong Base and Oxygen Scavenger % Change Ex. Time Absorbance from No.(days) System @ 600 nm Day 1 Observations 46 1 DI + 0.1 gm 0.893 SkyBlue, Na₂SO₃ +1 mL Significant NaOH (1N) Color Change 47 2 DI + 0.1 gm0.908 1.7 Sky Blue, Na₂SO₃ +1 mL Significant NaOH (1N) Color Change 48 6DI + 0.1 gm 1.020 14 Sky Blue, Na₂SO₃ +1 mL Significant NaOH (1N) ColorChange

[0107] TABLE 12 Stability of Pinacyanol Chloride in the Presence ofBuffer Ex. Time System¹ Absorbance % change No. (days) @ 600nm from Day1 Observations 49 1 Meta Buffer 1.038 Dark Purple, No Change 50 2 MetaBuffer 1.701 63.9 Precipitation 51 6 Meta Buffer 0.923 −11 Precipitation

[0108] TABLE 13 Stability of Pinacyanol Chloride in the Presence ofStrong Acid Ex. Time Absorbance @ % change No. (days) System 600 nm fromDay 1 Observations 52 1 DI + 2 drops H₂SO₄ Colorless (10N) 53 2 DI + 2drops H₂SO₄ Colorless (10N) 54 6 DI + 2 drops H₂SO₄ Colorless (10N)

[0109] TABLE 14 Stability of Pinacyanol Chloride in the Presence ofOxygen Scavenger Ex. Time Absorbance @ % change No. (days) System 600 nmfrom Day 1 Observations 55 1 DI + 0.1 gm 1.287 hydroquinone 56 6 DI +0.1 gm Light Brown, hydroquinone Significant Color Change

Examples 57-61

[0110] The stability of pinacyanol chloride, obtained from AldrichChemical Co., Milwaukee, Wis., in different solvent systems is tested,with all conditions being at room temperature, using a 1 inch (2.54 cm)pathlength cuvette, by dissolving the pinacyanol chloride in eachsolvent system, and recording the absorbance at 600 nm in a DR2000/2010Spectrophotometer supplied by HACH Company, Loveland, CO, after threeminutes mixing time. It is noted that where less than 100 vol %indicated for a solvent system, the balance was distilled water.Moreover, solubility issues led to the use of different concentrationsof the dye in the different solutions. Storage of the dye solutions isin amber bottles in air at 4° C. for the time indicated in the tables.Each absorbance was measured for a minimum of 6 samples, and the averagevalue is illustrated in the Table 15. TABLE 15 Stability of PinacyanolChloride in Solvent Systems as a function of time in days at 4° C. Ex.No. 57 58 59 60 61 Solvent Methanol Methanol Methyl Methyl Methyl (5 vol%) (100 Cellosolve Cellosolve Cellosolve vol %) (2 vol %) (5 vol %) (100vol %) Dye Conc. 8.93 × 1.79 × 9.0 × 9.0 × 1.8 × 10⁻⁵M 10⁻³M 10⁻³M 10⁻⁵M10⁻³M Time (days) Absorbance at 600 nm  1  1.489  1.469  1.439  1.445 1.445  6  1.426  1.462  1.364  1.365  1.419 20  1.421 22  1.446 %Change −4.2 −1.6 −5.2 −5.5 −1.7 from day 1

[0111] Although data shows promising results, it is noted that storageof the metachromatic dye compositions under refrigeration in the fieldis not as desirable as storage under ambient conditions.

Examples 62-63

[0112] The stability of pinacyanol chloride, obtained from AldrichChemical Co., Milwaukee, Wis., in distilled water, with purging with N₂gas in room temperature (20° C.) and low temperature, i.e., 4° C.,studies utilizing conditions as set forth in Examples 57-61. The resultsare shown in Table 16 TABLE 16 Stability of Pinacyanol Chloride inDistilled Water Using Nitrogen Purge At Room Temperature and 4° C. 62 63Ex. No. (20°) (4° C.) Time (days) Absorbance at 600 nm  1 1.463 1.463  71.392 1.466 23 1.446 % Change −4.8 −1.2 from day 1

[0113] Although data shows promising results, it is noted that storageof the metachromatic dye compositions under refrigeration in the fieldis not as desirable as storage under ambient conditions. Moreover,stabilization by purging the metachromatic dye composition after eachuse is not as desirable as stabilization with a technique that does notrequire repeated efforts on the part of field personnel.

EXAMPLE 64

[0114] Varying concentrations of pinacyanol chloride, obtained fromAldrich Chemical Co., Milwaukee, Wis., in a solution of 25 vol %methanol, Certified A.C.S. Grade Methanol with a purity of 99.8%obtained from Fischer Scientific, Pittsburgh, Pa., to 75 vol % ethyleneglycol, obtained from Fischer Scientific, Pittsburgh, Pa. (LaboratoryGrade indicated to be typically >99%, are prepared, and reacted with 0.2mg/L and 1.0 mg/L HPS-I, obtained from BetzDearborn Division of HerculesIncorporated, Trevose, Pa., with the visible absorbance being measuredin the same manner as in Example 12. The results are illustrated inTable 17. TABLE 17 Conc (M) Absorbance @ Absorbance @ PinacyanolChloride in 480 nm 480 nm 25% Methanol:75% Ethylene Glycol 0.2 mg/LHPS-I 1.0 mg/L HPS-I Linear Equation 1.07 × 10⁻⁴ 0.175 0.451 y =0.345x + 0.106 1.43 × 10⁻⁴ 0.236 0.487 y = 0.3138x + 0.1733 2.14 × 10⁻⁴0.309 0.604 y = 0.3688x + 0.2353 2.17 × 10⁻⁴ 0.318 0.621 y = 0.3788x +0.2423 2.91 × 10⁻⁴ 0.431 0.719 y = 0.36x + 0.359 3.58 × 10⁻⁴ 0.447 0.810y = 0.4538x + 0.3563

[0115] A plot of absorbance vs. concentration is plotted, as illustratedin FIG. 2. It is noted that as the concentration of the dye solutionincreases, the curves in FIG. 2 decrease. The slope of the curve isdetermined, and the optimum metachromatic dye concentration provides asmall change in the concentration of the HPS-I providing a large changein absorbance. This concentration provides a compromise between thesteepest slope and the smallest intercept, so that a small change in theconcentration of HPS-I in a water sample will correspond to a largechange in absorbance. Thus, a molar concentration of 2.17×10⁻⁴ is seento be a preferred concentration of the pinacyanol chloride. Inparticular, a dye concentration of 2.17×10⁻⁴M not only gives the highestsensitivity (as seen by the increase in the slope on the calibrationcurve in FIG. 2) but also produces the best accuracy in the calibrationrange (0.3 to 0.6 absorbance units for the recommended spectral rangefor a HACH DR2000/2010).

EXAMPLE 65

[0116] The stability of metachromatic dye compositions in sealed glassampoules was tested utilizing water purged with argon as follows.

[0117] The water used is distilled water which is purged thoroughly withargon to remove dissolved oxygen.

[0118] Three metachromatic dye compositions are prepared including ametachromatic dye concentration of 35 mg/L (9.0×10⁻⁵M) in the followingthree solvent systems:

[0119] a. 100% aqueous solution

[0120] b. 50% v/v water/propylene glycol

[0121] c. 50% v/v water/ethylene glycol.

[0122] The solutions were packaged in AccuVac ampoules, obtained fromHach Company, Loveland, Colo., and Voluette ampoules, obtained from HachCompany, Loveland, Colo., immediately after preparation. AccuVacampoules were filled with 1 mL of the dye solution, and the total volumeof the ampoule is 13.5 mL. The Voluette ampoules are filled with 2 mL ofthe dye solution, and the total volume of the ampoule is 2.2 mL.

[0123] The Voluette ampoules are amber in color. The AccuVac ampoulesare placed into styrofoam containers as soon as they were prepared toprotect them from light.

[0124] The packaging of the Voluette is under atmospheric pressure,whereas the AccuVac is under vacuum.

[0125] Results of the tests are shown in Table 18 for the metachromaticdye compositions, and in Table 19 for reaction with a HPS-I standardsolution.

[0126] With regard to testing the AccuVac ampoules, it is noted that asingle AccuVac ampoule gives vastly different absorbances depending onthe orientation in the cell holder. Absorbances varied from 0.307 to0.358 AU or 0.05 AU for the test which is ˜15% error. The reasons forthese significant variances include that the ampoules did not have aconsistent diameter, the ampoules were becoming stained with the dye,i.e., the dye precipitated, and imperfections in the quality of theglass.

[0127] The dye in the Voluette ampoules continued to show stabilityafter 2.5 months time. TABLE 18 Quality Check (@600 nm) of dye solutions(25 mL of DI water + 2 mL of dye solution) H₂O/Ethylene H₂O/PropyleneH₂O/Ethylene H₂O/Propylene H₂O Glycol Glycol Glycol Glycol SolventAccuVac AccuVac AccuVac Voluette Voluette Time (Days) Absorbance @ 600nm  1 1.587 1.722 1.782 1.736 1.707  4 1.301 1.479 1.519 1.731 1.691  61.147 1.400 1.414 1.633 1.664 20 1.622 1.674 82 1.625 1.663 % Change −28−19 −21 −6.4 2.6 from day 1

[0128] TABLE 19 Quality Check (@480 nm) of 25 mL of a 0.5 ppm HPS-Istandard solution + 1 mL Meta Buffer + 2 mL of dye solution H₂O/EthyleneH₂O/Propylene H₂O/Ethylene H₂O/Propylene Glycol Glycol Glycol GlycolTime AccuVac AccuVac Voluette Voluette 6 0.345 0.328 0.343 0.334 820.332 0.328

[0129] It is noted that the foregoing examples have been provided merelyfor the purpose of explanation and are in no way to be construed aslimiting of the present invention. While the present invention has beendescribed with reference to an exemplary embodiment, it is understoodthat the words which have been used herein are words of description andillustration, rather than words of limitation. Changes may be made,within the purview of the appended claims, as presently stated and asamended, without departing from the scope and spirit of the presentinvention in its aspects. Although the present invention has beendescribed herein with reference to particular means, materials andembodiments, the present invention is not intended to be limited to theparticulars disclosed herein; rather, the present invention extends toall functionally equivalent structures, methods and uses, such as arewithin the scope of the appended claims.

What is claimed is:
 1. A stabilized metachromatic dye composition havinga percent change in absorbance of less than about 10% when stored for aperiod of about one week.
 2. The stabilized metachromatic dyecomposition according to claim 1 wherein the percent change inabsorbance is less than about 1% when stored for a period of about oneweek.
 3. The stabilized metachromatic dye composition according to claim1 wherein the percent change in absorbance is less than about 3% whenstored for a period of about one month.
 4. The stabilized metachromaticdye composition according to claim 1 wherein the percent change inabsorbance is less than about 5% when stored for a period of about 3months.
 5. The stabilized metachromatic dye composition according toclaim 1 wherein the percent change in absorbance is less than about 5%when stored for a period of about 6 months.
 6. The stabilizedmetachromatic dye composition according to claim 5 comprising an aqueoussolution of metachromatic dye.
 7. The stabilized metachromatic dyecomposition according to claim 5 comprising a non-aqueous solution ofmetachromatic dye.
 8. The stabilized metachromatic dye compositionaccording to claim 1 wherein the percent change in absorbance is lessthan about 10% when stored for a period of about one year.
 9. Thestabilized metachromatic dye composition according to claim 8 comprisingan aqueous solution of metachromatic dye.
 10. The stabilizedmetachromatic dye composition according to claim 8 comprising anon-aqueous solution of metachromatic dye.
 11. The stabilizedmetachromatic dye composition according to claim 1 wherein the percentchange in absorbance is less than about 5% when stored for a period ofabout one year.
 12. The stabilized metachromatic dye compositionaccording to claim 11 comprising an aqueous solution of metachromaticdye.
 13. The stabilized metachromatic dye composition according to claim11 comprising a non-aqueous solution of metachromatic dye.
 14. Anaqueous solution of metachromatic dye comprising metachromatic dye in anaqueous solvent, said aqueous solvent having a metachromatic dyestabilizing pH.
 15. The aqueous solution of metachromatic dye accordingto claim 14 wherein said aqueous solvent has a pH of a least about 8.16. The aqueous solution of metachromatic dye according to claim 15wherein said aqueous solvent has a pH of a least about
 10. 17. Theaqueous solution of metachromatic dye according to claim 16 wherein saidaqueous solvent has a pH of a least about
 11. 18. The aqueous solutionof metachromatic dye according to claim 14 wherein said aqueous solventhas a pH of about 8 to
 14. 19. The aqueous solution of metachromatic dyeaccording to claim 18 wherein said aqueous solvent has a pH of about 11to
 12. 20. The aqueous solution of metachromatic dye according to claim19 wherein said aqueous solvent has a pH of about 11 to 11.5.
 21. Theaqueous solution of metachromatic dye according to claim 14 wherein saidmetachromatic dye comprises at least one of pinacyanol chloride, crystalviolet, methyl green, malachite green, acridin orange, paraosaniline,nile blue A, neutral red, safrin O, methylene blue, methyl red,brilliant green, toluidine blue, new methylene blue, quinalizarin,tetrachrome, brilliant blue G, and mordant black II.
 22. The aqueoussolution of metachromatic dye according to claim 21 wherein saidmetachromatic dye comprises pinacyanol chloride.
 23. The aqueoussolution of metachromatic dye according to claim 14 including at leastone basic material.
 24. The aqueous solution of metachromatic dyeaccording to claim 23 wherein said at least one basic material comprisesa buffer.
 25. The aqueous solution of metachromatic dye according toclaim 23 wherein said at least one basic material comprises at least oneof sodium hydroxide, potassium hydroxide and lithium hydroxide.
 26. Theaqueous solution of metachromatic dye according to claim 25 wherein saidat least one basic material comprises sodium hydroxide.
 27. The aqueoussolution of metachromatic dye according to claim 22 wherein said atleast one basic material comprises sodium hydroxide.
 28. The aqueoussolution of metachromatic dye according to claim 14 further including atleast one non-aqueous solvent.
 29. The aqueous solution of metachromaticdye according to claim 14 further including at least one oxygenscavenger.
 30. The aqueous solution of metachromatic dye according toclaim 14 wherein the aqueous solution is substantially free of oxygen.31. A metachromatic dye solution comprising metachromatic dye andnon-aqueous solvent, the solution being substantially free of water. 32.The metachromatic dye solution according to claim 31 wherein saidmetachromatic dye comprises at least one of pinacyanol chloride, crystalviolet, methyl green, malachite green, acridin orange, paraosaniline,nile blue A, neutral red, safrin O, methylene blue, methyl red,brilliant green, toluidine blue, new methylene blue, quinalizarin,tetrachrome, brilliant blue G, and mordant black II.
 33. Themetachromatic dye solution according to claim 32 wherein saidmetachromatic dye comprises pinacyanol chloride.
 34. The metachromaticdye solution according to claim 31 wherein said non-aqueous solventcomprises at least one of alcohols, methylcellosolve, hexane, pentane,heptane, toluene, xylene, benzene, dichlorobenzene, acetone, ethylacetate, diethyl ether, acetonitrile, dimethylsulfoxide.
 35. Themetachromatic dye solution according to claim 31 wherein saidnon-aqueous solvent comprises at least one of methanol, ethanol,butanol, isopropanol, propanol and ethylene glycol.
 36. Themetachromatic dye solution according to claim 31 wherein saidnon-aqueous solvent has a density at 25° C. of about 0.95 to 1.2 g/cm³.37. The metachromatic dye solution according to claim 36 wherein saidnon-aqueous solvent has a density at 25° C. of about 1 to 1.1 g/cm³. 38.The metachromatic dye solution according to claim 37 wherein saidnon-aqueous solvent has a density at 25° C. of about 1 to 1.05 g/cm³.39. The metachromatic dye solution according to claim 31 wherein saidnon-aqueous solvent comprises a mixture of non-aqueous solvents.
 40. Themetachromatic dye solution according to claim 39 wherein said mixture ofnon-aqueous solvents comprises a mixture of methanol and ethyleneglycol.
 41. The metachromatic dye solution dye according to claim 39wherein said mixture of non-aqueous solvents comprises a mixturecontaining 25 vol % methanol and 75 vol % ethylene glycol.
 42. Themetachromatic dye solution according to claim 36 wherein saidnon-aqueous solvent comprises a mixture of non-aqueous solvents.
 43. Themetachromatic dye solution according to claim 42 wherein said mixture ofnon-aqueous solvents comprises a mixture of methanol and ethyleneglycol.
 44. The metachromatic dye solution according to claim 43 whereinsaid metachromatic dye comprises pinacyanol chloride.
 45. Themetachromatic dye solution according to claim 31 wherein the solution isfree of water.
 46. The metachromatic dye solution dye according to claim31 wherein said non-aqueous solvent comprises high purity solvent. 47.The metachromatic dye solution according to claim 31 further includingan oxygen scavenger.
 48. The metachromatic dye solution according toclaim 31 in the substantial absence of oxygen.
 49. The metachromatic dyesolution according to claim 31 wherein the solution is purged with aninert gas.
 50. A container including therein the aqueous solution ofmetachromatic dye recited in claim
 14. 51. The container according toclaim 50, wherein the container is at least reduces the transmission oflight.
 52. The container according to claim 51, wherein the containerprevents the transmission of light.
 53. A container including thereinthe aqueous solution of metachromatic dye recited in claim
 19. 54. Thecontainer according to claim 53, wherein the container is at leastreduces the transmission of light.
 55. The container according to claim54, wherein the container prevents the transmission of light.
 56. Acontainer including therein the solution of metachromatic dye recited inclaim
 31. 57. The container according to claim 56, wherein the containeris at least reduces the transmission of light.
 58. The containeraccording to claim 57, wherein the container prevents the transmissionof light.
 59. A container including therein the solution ofmetachromatic dye recited in claim
 36. 60. The container according toclaim 59, wherein the container is at least reduces the transmission oflight.
 61. The container according to claim 60, wherein the containerprevents the transmission of light.
 62. A container including thereinthe solution of metachromatic dye recited in claim
 42. 63. The containeraccording to claim 62, wherein the container is at least reduces thetransmission of light.
 64. The container according to claim 63, whereinthe container prevents the transmission of light.
 65. A containerincluding therein the solution of metachromatic dye recited in claim 45.66. The container according to claim 65, wherein the container is atleast reduces the transmission of light.
 67. The container according toclaim 66, wherein the container prevents the transmission of light.